Evaluation of cytotoxic activity of titanocene difluorides and determination of their mechanism of action in ovarian cancer cells


Background Ovarian cancer is the seventh-most common cancer amongst women and the most deadly gynecologic cancer. Cisplatin based drugs are used in first line therapy, but resistance represents a major obstacle for successful treatment. In this study, we investigated the anticancer effects and mechanism of action of three titanocene difluorides, two bearing a pendant carbohydrate moiety (α-d-ribofuranos-5-yl) on their periphery and one without any substitution. Results The efficacy of these compounds on ovarian cancer cell lines was evaluated in relation to their particular chemical structure and compared with cisplatin as the most common treatment modality for this type of cancer. The typical mechanism of cisplatin action involves DNA damage, activation of p53 protein and induction of cell death, as previously described for titanium ions. Nevertheless, our data indicate that the effect of titanocene difluoride derivatives is mediated via the endoplasmic reticulum stress pathway and autophagy. Conclusion We anticipate that the presence of substituents on cyclopentadienyl ring(s) might play an important role in modulation of the activity of particular compounds. Titanocene difluorides exert comparable cytotoxic activity as cisplatin and are more efficient in cisplatin-resistant cell lines. Our results suggest potential utilization of these compounds especially in the treatment of cisplatin-resistant tumor cells.

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  1. 1.

    Kopf H, Kopf-Maier P (1979) Titanocene dichloride—the first metallocene with cancerostatic activity. Angew Chem Int Ed Engl 18:477–478

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Ana M, Pizarro AH, Sadler PJ (2010) Activation mechanisms for organometallic anticancer complexes. Top Organomet Chem 32:21–56

    Google Scholar 

  3. 3.

    Lummen G, Sperling H, Luboldt H, Otto T, Rubben H (1998) Phase II trial of titanocene dichloride in advanced renal-cell carcinoma. Cancer Chemother Pharmacol 42:415–417

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Deally A, Hackenberg F, Lally G, Tacke M (2012) Synthesis and biological evaluation of achiral indole-substituted titanocene dichloride derivatives. Int J Med Chem. doi:10.1155/2012/905981

    PubMed Central  PubMed  Google Scholar 

  5. 5.

    Gasser G, Ott I, Metzler-Nolte N (2011) Organometallic anticancer compounds. J Med Chem 54:3–25

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  6. 6.

    Hearn JM, Romero-Canelon I, Qamar B, Liu Z, Hands-Portman I, Sadler PJ (2013) Organometallic Iridium(III) anticancer complexes with new mechanisms of action: NCI-60 screening, mitochondrial targeting, and apoptosis. ACS Chem Biol 8:1335–1343

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  7. 7.

    Olszewski U, Claffey J, Hogan M, Tacke M, Zeillinger R, Bednarski PJ, Hamilton G (2011) Anticancer activity and mode of action of titanocene C. Investig New Drugs 29:607–614

    CAS  Article  Google Scholar 

  8. 8.

    Meier SM, Novak M, Kandioller W, Jakupec MA, Arion VB, Metzler-Nolte N, Keppler BK, Hartinger CG (2013) Identification of the structural determinants for anticancer activity of a ruthenium arene peptide conjugate. Chemistry 19:9297–9307

    CAS  Article  PubMed  Google Scholar 

  9. 9.

    Gómez-Ruiz S, Maksimović-Ivanić D, Mijatović S, Kaluđerović GN (2012) On the discovery, biological effects, and use of cisplatin and metallocenes in anticancer chemotherapy. Bioinorg Chem Appl. doi:10.1155/2012/140284

    Google Scholar 

  10. 10.

    Eger S, Immel TA, Claffey J, Muller-Bunz H, Tacke M, Groth U, Huhn T (2010) Titanocene difluorides with improved cytotoxic activity. Inorg Chem 49:1292–1294

    CAS  Article  PubMed  Google Scholar 

  11. 11.

    Koleros E, Stamatatos TC, Psycharis V, Raptopoulou CP, Perlepes SP, Klouras N (2010) In search for titanocene complexes with improved cytotoxic activity: synthesis, x-ray structure, and spectroscopic study of Bis(eta-cyclopentadienyl)difluorotitanium(IV). Bioinorg Chem Appl. doi:10.1155/2010/914580

    PubMed Central  PubMed  Google Scholar 

  12. 12.

    Guo M, Sun H, McArdle HJ, Gambling L, Sadler PJ (2000) Ti(IV) uptake and release by human serum transferrin and recognition of Ti(IV)-transferrin by cancer cells: understanding the mechanism of action of the anticancer drug titanocene dichloride. Biochemistry 39:10023–10033

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Pavlaki M, Debeli K, Triantaphyllidou I-E, Klouras N, Giannopoulou E, Aletras A (2009) A proposed mechanism for the inhibitory effect of the anticancer agent titanocene dichloride on tumour gelatinases and other proteolytic enzymes. J Biol Inorg Chem 14:947–957

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Bharti SK, Singh SK (2009) Recent developments in the field of anticancer metallopharmaceuticals. Int J Pharm Technol Res 1:1406–1420

    CAS  Google Scholar 

  15. 15.

    Hodík T, Lamač M, Červenková Št’astná L, Karban J, Koubková L, Hrstka R, Císařová I, Pinkas J (2014) Titanocene dihalides and ferrocenes bearing a pendant α-D-Xylofuranos-5-yl or α-D-Ribofuranos-5-yl Moiety. Synthesis, characterization, and cytotoxic activity. Organometallics 33:2059–2070

    Article  Google Scholar 

  16. 16.

    Blaydes JP, Hupp TR (1998) DNA damage triggers DRB-resistant phosphorylation of human p53 at the CK2 site. Oncogene 17:1045–1052

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Kvardova V, Hrstka R, Walerych D, Muller P, Matoulkova E, Hruskova V, Stelclova D, Sova P, Vojtesek B (2010) The new platinum(IV) derivative LA-12 shows stronger inhibitory effect on Hsp90 function compared to cisplatin. Mol Cancer. doi:10.1186/1476-4598-9-147

    PubMed Central  PubMed  Google Scholar 

  18. 18.

    Vojtesek B, Bartek J, Midgley CA, Lane DP (1992) An immunochemical analysis of the human nuclear phosphoprotein p53. New monoclonal antibodies and epitope mapping using recombinant p53. J Immunol Methods 151:237–244

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Fredersdorf S, Milne AW, Hall PA, Lu X (1996) Characterization of a panel of novel anti-p21Waf1/Cip1 monoclonal antibodies and immunochemical analysis of p21Waf1/Cip1 expression in normal human tissues. Am J Pathol 148:825–835

    PubMed Central  CAS  PubMed  Google Scholar 

  20. 20.

    Nguyen HL, Zucker S, Zarrabi K, Kadam P, Schmidt C, Cao J (2011) Oxidative stress and prostate cancer progression are elicited by membrane-type 1 matrix metalloproteinase. Mol Cancer Res 9:1305–1318

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  21. 21.

    Christodoulou CV, Eliopoulos AG, Young LS, Hodgkins L, Ferry DR, Kerr DJ (1998) Anti-proliferative activity and mechanism of action of titanocene dichloride. Br J Cancer 77:2088–2097

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  22. 22.

    Blaydes JP, Craig AL, Wallace M, Ball HM, Traynor NJ, Gibbs NK, Hupp TR (2000) Synergistic activation of p53-dependent transcription by two cooperating damage recognition pathways. Oncogene 19:3829–3839

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Kopf-Maier P, Wagner W, Liss E (1983) Induction of cell arrest at G1/S and in G2 after treatment of Ehrlich ascites tumor cells with metallocene dichlorides and cis-platinum in vitro. J Cancer Res Clin Oncol 106:44–52

    CAS  Article  PubMed  Google Scholar 

  24. 24.

    Jungwirth U, Kowol CR, Keppler BK, Hartinger CG, Berger W, Heffeter P (2011) Anticancer activity of metal complexes: involvement of redox processes. Antioxid Redox Signal 15:1085–1127

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  25. 25.

    Peng X, Gandhi V (2012) ROS-activated anticancer prodrugs: a new strategy for tumor-specific damage. Ther Deliv 3:823–833

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  26. 26.

    Marullo R, Werner E, Degtyareva N, Moore B, Altavilla G, Ramalingam SS, Doetsch PW (2013) Cisplatin induces a mitochondrial-ROS response that contributes to cytotoxicity depending on mitochondrial redox status and bioenergetic functions. PLoS One. doi:10.1371/journal.pone.0081162

    Google Scholar 

  27. 27.

    Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, Murakami T, Taniguchi M, Tanii I, Yoshinaga K, Shiosaka S, Hammarback JA, Urano F, Imaizumi K (2006) Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol 26:9220–9231

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  28. 28.

    Sano R, Reed JC (2013) ER stress-induced cell death mechanisms. Biochim Biophys Acta 1833:3460–3470

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Yorimitsu T, Nair U, Yang Z, Klionsky DJ (2006) Endoplasmic reticulum stress triggers autophagy. J Biol Chem 281:30299–30304

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  30. 30.

    Yin JJ, Li YB, Wang Y, Liu GD, Wang J, Zhu XO, Pan SH (2012) The role of autophagy in endoplasmic reticulum stress-induced pancreatic beta cell death. Autophagy 8:158–164

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Schonthal AH (2012) Endoplasmic reticulum stress: its role in disease and novel prospects for therapy. Scientifica. doi:10.6064/2012/857516

    PubMed Central  PubMed  Google Scholar 

  32. 32.

    Sponer JE, Leszczynski J, Sponer J (2006) Mechanism of action of anticancer titanocene derivatives: an insight from quantum chemical calculations. J Phys Chem B 110:19632–19636

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Chen X, Zhou L (2010) The hydrolysis chemistry of anticancer drug titanocene dichloride: an insight from theoretical study. J Mol Struct THEOCHEM 940:45–49

    CAS  Article  Google Scholar 

  34. 34.

    Ravera M, Cassino C, Monti E, Gariboldi M, Osella D (2005) Enhancement of the cytotoxicity of titanocene dichloride by aging in organic co-solvent. J Inorg Biochem 99:2264–2269

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Zhang Z, Yang P, Guo M, Wang H (1996) Effect of titanocene dichloride coordination on Watson-Crick base pairing. J Inorg Biochem 63:183–190

    CAS  Article  Google Scholar 

  36. 36.

    Vandewynckel YP, Laukens D, Geerts A, Bogaerts E, Paridaens A, Verhelst X, Janssens S, Heindryckx F, Van Vlierberghe H (2013) The paradox of the unfolded protein response in cancer. Anticancer Res 33:4683–4694

    CAS  PubMed  Google Scholar 

  37. 37.

    Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, Rosen J, Eskelinen EL, Mizushima N, Ohsumi Y, Cattoretti G, Levine B (2003) Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 112:1809–1820

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  38. 38.

    Yang ZJ, Chee CE, Huang S, Sinicrope FA (2011) The role of autophagy in cancer: therapeutic implications. Mol Cancer Ther 10:1533–1541

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  39. 39.

    Vinod V, Padmakrishnan CJ, Vijayan B, Gopala S (2014) ‘How can I halt thee?’ The puzzles involved in autophagic inhibition. Pharmacol Res Off J Ital Pharmacol Soc 82:1–8

    CAS  Google Scholar 

  40. 40.

    Vlahopoulos S, Critselis E, Voutsas IF, Perez SA, Moschovi M, Baxevanis CN, Chrousos GP (2014) New use for old drugs? Prospective targets of chloroquines in cancer therapy. Curr Drug Targets 15:843–851

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Tanida I, Ueno T, Kominami E (2008) LC3 and autophagy. Methods Mol Biol 445:77–88

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Zhang XJ, Chen S, Huang KX, Le WD (2013) Why should autophagic flux be assessed? Acta Pharmacol Sin 34:595–599

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  43. 43.

    Winterbourn CC (1995) Toxicity of iron and hydrogen peroxide: the Fenton reaction. Toxicol Lett 82–83:969–974

    Article  PubMed  Google Scholar 

  44. 44.

    Bolisetty S, Jaimes EA (2013) Mitochondria and reactive oxygen species: physiology and pathophysiology. Int J Mol Sci 14:6306–6344

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  45. 45.

    Ray PD, Huang BW, Tsuji Y (2012) Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 24:981–990

    PubMed Central  CAS  Article  PubMed  Google Scholar 

  46. 46.

    Halliwell B (2007) Biochemistry of oxidative stress. Biochem Soc Trans 35:1147–1150

    CAS  Article  PubMed  Google Scholar 

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We would like to thank Dr. Philip J. Coates for critical reading of the manuscript. This work was supported by the project MEYS – NPS I – LO1413, MH CZ-DRO (MMCI, 00209805) and Czech Science Foundation projects P206/12/G151 and P207/12/2368.

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The authors declare no conflict of interest.

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Correspondence to Roman Hrstka.

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Koubkova, L., Vyzula, R., Karban, J. et al. Evaluation of cytotoxic activity of titanocene difluorides and determination of their mechanism of action in ovarian cancer cells. Invest New Drugs 33, 1123–1132 (2015). https://doi.org/10.1007/s10637-015-0274-y

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  • Titanocene
  • Cisplatin
  • Ovarian cancer
  • Cytotoxicity
  • Organometallic compounds